Compositions and methods for treatment of hereditary cystatin c amyloid angiopathy (hccaa) and other neurodegenerative disorders associated with aberrant amyloid deposits

ABSTRACT

Compositions and Methods for the Treatment of Amyloid Deposit diseases, e.g., Hereditary cystatin C amyloid angiopathy and other neurodegenerative disorders, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/789,923, filed Feb. 13, 2020, which is a continuation ofU.S. patent application Ser. No. 16/124,798, filed Sep. 7, 2018, whichclaims priority to U.S. Provisional Application No. 62/555,496 filedSep. 7, 2017, the entire contents being incorporated herein by referenceas though set forth in full.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Incorporated herein by reference in its entirety is the Sequence Listingsubmitted via EFS-Web as a text file named 6517US03_SequenceListing.txt,created May 20, 2022 and having a size of 1,280 bytes.

FIELD OF THE INVENTION

The present invention relates to the fields of angiopathy, most notablyincluding cerebral amyloid angiopathy and neurodegenerative disorders,associated with pathogenic fibril formation. More specifically theinvention provides compositions and methods useful for the treatment andmanagement of diseases associated with aberrant fibril formation,particularly hereditary cystatin C amyloid angiopathy (HCCAA) andAlzheimer's disease.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited through thespecification in order to describe the state of the art to which thisinvention pertains. Each of these citations is incorporated herein byreference as though set forth in full.

Hereditary cystatin C amyloid angiopathy (HCCAA) is a dominantlyinherited disease caused by a leucine 68 to glutamine variant of humancystatin C (hCC; L68Q-hCC).¹ HCCAA is classified as a cerebral amyloidangiopathy (CAA), a group of diseases in which amyloid deposits form onthe walls of blood vessels in the central nervous system (CNS). AlthoughHCCAA is rightly classified as a CAA disorder due to its strong cerebralpresentation, hCC deposition is systemic and is also found in otherinternal organs. Most carriers of the mutation suffer micro-infarcts andbrain hemorrhages in their twenties leading to paralysis, dementia anddeath in young adults, with an average life expectancy of 30 years.²⁻⁶Post-mortem studies in humans show that hCC is deposited in all brainareas, grey and white matter alike, most prominently in arteries andarterioles.

Human cystatin C, a cysteine protease inhibitor that belongs to thecystatin super-family, is a secretory type 2 cystatin, expressed in allnucleated human cells and found in all tissues and body fluids and atparticularly high concentrations in cerebrospinal fluid.^(2, 7-9) hCCinhibits cysteine proteases like papain and legumain by its interactionthrough multiple binding motifs resulting from the characteristic hCCfold.⁹⁻¹¹ Its normal conformation is composed of a polypeptide thatfolds into a five-stranded β-sheet, which partially wraps around acentral α-helix. The N-terminal segment and two hair-pin loops build theedge of the protein, which binds into the active site of cysteineproteases and blocks their proteolytic activity.¹²⁻¹⁴ The mutation ofleucine 68 to glutamic acid destabilizes the packing between the betasheets and the alpha helix, allowing the molecule to open. Two such openhCC molecules can interact with each other, with the helix of eachmolecule interacting with the beta sheet of the other; the resultingdimer is said to be the product of domain swapping.¹⁵⁻¹⁷ Additionally,through a process called propagated domain swapping, long chains ofmolecules can be built, in which the free domain of each moleculeinteracts with a new hCC monomer.¹⁸ The aggregation of proteins leads tothe formation of highly ordered pathogenic fibrillar aggregates, calledamyloid fibrils,^(19, 20) which are implicated not only in HCCAA butalso in a wide range of neurodegenerative diseases such as Alzheimer's,Parkinson's, Creutzfeldt-Jacob's, Huntington's disease and other CAAs.²⁰

The degree of amyloid maturation observed in cystatin C deposits hasbeen shown to vary between tissues (i.e, less prominent maturation inskin than in brain).²¹ Although deposits in the skin are not comprisedof amyloid fibers, quantitative studies on hCC deposition within theskin of mutant carriers showed that symptomatic carriers hadsignificantly higher levels of hCC immunoreactivity in their skin thanasymptomatic carriers. The fact that the quantity of hCC deposition inskin was associated with the progression of the disease in the CNS showsthat skin biopsies could be used to assess disease progression andcould, therefore, be of use in the evaluation of therapeuticinterventions.²²

Protein oligomers of different pathogenic amyloidogenic proteins precedethe fibril formation stage in HCCAA and other diseases, although forHCCAA it is unclear if such oligomers lead directly to pathogenicfibrils, or if assembly of fibrils occurs most rapidly from monomers.²³Drugs reducing aggregation of amyloid-producing proteins have thepotential to reduce the formation of toxic oligomers known to occur inseveral types of amyloidosis.^(24, 25) Previous investigations havesuggested that preventing domain swapping of hCC might be used fortreatment of HCCAA;²⁴ Nilsson and colleagues developed variants of WThCC and L6Q-hCC with intra-chain stabilizing disulfide bonds preventingdomain swapping that could form either dimers or amyloid fibrils.²⁶These results suggest that the knowledge of the molecular mechanismcausing the transition of physiologically normal and soluble proteins totoxic oligomers and insoluble fibrils is essential for the developmentof treatment strategies.

Östner et al. have previously attempted to prevent polymerization of hCCmonomers, or disrupt or remove multimeric species, through variousapproaches.²⁴ As mentioned, modified, stabilized hCC monomers have beenused to demonstrate that preventing domain swapping preventsaggregations. Antibodies can be raised specifically against the domainswapped, dimeric form of hCC; those antibodies were able to specificallyremove dimers of hCC, and not monomers, from patient plasma using sizeexclusion chromatography.²⁷ A high-throughput screen of compounds hasbeen pursued using the US Drug Collection (comprised of 1040 FDAapproved compounds; (found on the world wide web at.msdiscovery.com/usdrug.html) in an effort to find molecules thatprevent dimerization.²⁴ Although promising, this approach required largeamounts of purified hCC protein produced in bacteria, and the compoundsthat were identified as inhibiting dimer formation were for the mostpart used at concentrations too high to be considered therapeutic in anorganism.

Clearly, there is a need for improved methods and compositions fortreating HCCAA.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method for treating amyloiddeposit disease comprising delivering an effective amount of at leastone antioxidant to a patient, said anti-oxidant disrupting said amyloiddeposit, thereby alleviating disease symptoms. Amyloid deposit diseases,include for example, hereditary cystatin C amyloid angiopathy (HCCAA),Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jacob's disease,Huntington disease and other forms of cerebral amyloid angiopathies suchas the Dutch form of the disease. In certain embodiments, the amyloiddisease is HCCAA caused by mutated cystatin C. In other embodiments, themutated cystatin C comprises a L68Q cystatin C. Preferred antioxidantsfor use in the method above, include, without limitation, glutathione,N-acetyl cysteine or a derivative thereof. In certain embodiments, thederivatives are selected from NAC-amide, NAC-ethyl ester and zincmercaptide N-acetyl cysteine carboxylate salt.

In another aspect, a method for treatment of hereditary cystatin Camyloid angiopathy (HCCAA) in a human subject in need thereof isprovided. An exemplary method comprises administration of an effectiveamount of N-acetyl cysteine or functional derivative thereof in apharmaceutically acceptable carrier to the subject, the administrationbeing effective to reduce amyloid-cystatin protein aggregates, therebyalleviating symptoms of HCCAA. In certain embodiments, the NACderivative is selected from NAC-amide, NAC-ethyl ester and zincmercaptide N-acetyl cysteine carboxylate salt. The method can optionallyentail performing a skin biopsy on said subject following treatment toassess reduction in amyloid-cystatin protein aggregates in skin ormeasuring cystatin C monomer, dimer or oligomer in serum or plasma orthe amount of monomer excreted in the urine.

In another aspect, the method can entail administration of additionalagents which alleviate amyloid deposit symptoms. These include withoutlimitation, one or more ionophores, one or more anti-inflammatory agentsand one or more proteases. In other embodiments, siRNA directed tocystatin C coding sequences are administered to selectively block themutated allele.

In another aspect of the invention, a method for treatment of aneurodegenerative disorder associated with pathogenic fibrillationprotein aggregates in a human subject in need thereof is disclosed. Anexemplary method comprising administration of an effective amount ofN-acetyl cysteine or functional derivative thereof in a pharmaceuticallyacceptable carrier to the subject wherein the administration iseffective to reduce said protein fibril aggregates, thereby alleviatingsymptoms of the neurodegenerative disorder. In certain embodiments, thedisorder is selected from Alzheimer's disease, Parkinson's disease,Creutzfeldt-Jacob's disease, Huntington disease and other forms ofcerebral amyloid angiopathy (CAA) such as the Dutch form.

In certain embodiments, the methods above comprise monitoring saidpatient for amyloid deposit levels.

In yet another aspect of the invention, a method for identifyingtherapeutic agents which alter amyloid-cystatin protein aggregateformation is provided. An exemplary method comprising providing cellsexpressing a nucleic acid encoding a mutant hCC protein, said mutantcausing formation of amyloid-cystatin protein aggregates; and providingcells which express a hCC protein which lacks the hCC mutation. Bothpopulations of cells are contacted with a test agent and assessed todetermine whether the agent alters amyloid-cystatin protein aggregateformation of cells expressing the mutant relative to those expressingthe wild type protein, thereby identifying agents which alteramyloid-cystatin protein aggregation. Agents so identified should haveefficacy for the treatment of HCCAA or other disorders associatedaberrant fibril formation.

Also provided is a pharmaceutical composition comprising an effectiveamount of an agent which acts as an antioxidant and, or a reducing agentfor the treatment of amyloid deposit disease in a pharmaceuticallyacceptable carrier. Diseases to be treated with the composition, includefor example, HCCAA, Alzheimer's disease, Parkinson's disease,Creutzfeldt-Jacob's disease, Huntington disease and other CAAs. In oneembodiment, the agent is glutathione, N-acetyl cysteine or a derivativethereof. In a preferred embodiment, the agent is a derivative and isselected from NAC-amide, NAC-ethyl ester and zinc mercaptide N-acetylcysteine carboxylate salt. The inventive compositions of the inventioncan also comprise one or more of an ionophore, an anti-inflammatoryagent or a protease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Genetically engineered HEK-293T cells produce and secretedetectable levels of hCC (WT or L68Q) capable of oligomerizing undernon-reducing conditions. FIG. 1A. Schematic representation of WT andL68Q mutant hCC proteins. Dashed line represents the N-terminal signalpeptide subject to proteolysis. The red rectangle represents the Myc tagadded to the C-terminal end. FIG. 1B. (left panel) Lysates from HEK-293Tcells stably expressing hCC WT or L68Q mutant or supernatants (rightpanel) were mixed with 2% SDS with or without the reducing agents DTT orβ-mercaptoetanol when indicated. Samples were subject to electrophoresisand CST3 levels examined by the Western blot procedure usinganti-cystatin C antibody. FIG. 1B: Incubation with glutathione impairscystatin C di/oligomerization in cellular extracts and supernatants(biological replica) FIG. 1C. Biological replicates related toexperiments shown in FIG. 2 where supernatants and cellular extractswere incubated in the presence of glutathione during 1 h at 37° C. withindicated concentrations. Samples were mixed with 2% SDS withoutreducing agents prior to electrophoresis, and proteins levels weredetected by anti-cystatin C antibody WB.

FIG. 2A: Incubation with glutathione impairs cystatin Cdi/oligomerization in cellular extracts and supernatants. Supernatantsand cellular extracts were incubated in the presence of glutathione atthe indicated concentrations for 1 h at 37° C. Samples were mixed with2% SDS without reducing agents prior to electrophoresis, and proteinlevels were detected by Western blot for cystatin C. (N=3; * significantat P<0.05 with respect to untreated (HMW); +significant at P<0.05 withrespect to untreated (monomer)). FIG. 2B: Glutathione andN-acetylcysteine impairs oligomerization of secreted cystatin C L68Q(Biological replicates). Biological replicates related to experimentsshown in FIG. 3 where supernatants were incubated in the presence ofglutathione or NAC during 1 h at 37° C. with indicated concentrations.Samples were mixed with 2% SDS without reducing agents prior toelectrophoresis, and proteins levels were detected by anti-cystatin Cantibody WB.

FIG. 3A: Glutathione and N-acetylcysteine impairs oligomerization ofsecreted cystatin C L68Q. Supernatants were incubated in the presence ofthe indicated concentrations of glutathione or NAC for 1 h at 37° C.Samples were mixed with 2% SDS without reducing agents prior toelectrophoresis, and protein levels were detected by anti-cystatin Cantibody. The histogram represents the quantification by densitometry ofthe Western blot bands for the high molecular weight fraction (HMW)relative to the untreated sample or monomer (Mono) relative to the DTTtreated sample. No HMW fraction was detected in supernatants fromHEK-293T cells stably expressing hCC WT. (* significant at P<0.05 withrespect to untreated (HMW); +significant at P<0.05 with respect tountreated (Monomer)). FIG. 3B: N-acetylcysteine impairs oligomerizationof secreted cystatin C L68Q (Biological replicates) Biologicalreplicates related to experiments shown in FIG. 4 where 293T cellsexpressing WT or L68Q cystatin C were incubated with the indicatedamounts of compound during 24, 48 or 72 h. Small amounts of supernatantwere removed from the cells and analyzed by Western blot at theindicated times. On days 2 and 3, only the L68Q supernatants wereanalyzed. Samples were mixed with 2% SDS without reducing agents priorto electrophoresis, and protein levels were detected by anti-cystatin Cantibody WB.

FIG. 4: NAC impairs oligomerization of secreted hCC L68Q. 293T cellsexpressing WT or L68Q cystatin C were incubated with media containingthe indicated amount of either GSH or NAC for 24, 48 or 72 h. Smallamounts of supernatant were removed from the cells and analyzed byWestern blot at the indicated times. On days 2 and 3, only thesupernatants from cells expressing hCC L68Q variant were analyzed.Samples were mixed with 2% SDS without reducing agents prior toelectrophoresis, and protein levels were detected by anti-cystatin Cantibody. The histogram represents the quantification by densitometry ofthe Western blot bands for the high molecular weight fraction (HMW)relative to the untreated sample or monomer (Mono) relative to the DTTtreated sample. (* significant at P<0.05 with respect to untreated(HMW); +significant at P<0.05 with respect to untreated (Monomer)).

FIG. 5. Reducing activity of GSH or NAC are critical for breakingoligomers into monomers of secreted Cystatin C L68Q. Supernatants wereincubated in presence of oxidized (GSSG) or reduced glutathione (GSH),NAC or its inactive analogous (NAS) during 1 h at 37° C. with indicatedconcentrations. Samples were mixed with 2% SDS without reducing agentsprior to electrophoresis, and proteins levels were detected byanti-cystatin C antibody.

FIG. 6. NAC-amide and NAC-ethyl-ester impair oligomerization ofintracellular and secreted Cystatin C L68. Supernatants and cellularextracts were incubated in presence of NAC, NAC-amide and NAC-methylester during 1 h at 37C with indicated concentrations. Samples weremixed with 2% SDS without reducing agents prior to electrophoresis, andproteins levels were detected by anti-cystatin C antibody.

FIG. 7. High molecular weight complexes of Cyst-C L68Q can be detectedin transgenic mice. Short incubation of NAC impairs oligomerization ofCyst-C L68Q on blood and brain extracts. Plasma or brain extracts wereincubated in presence of NAC during 1 h at 37° C. with indicatedconcentrations. Samples were mixed with 2% SDS without reducing agentsprior to electrophoresis, and proteins levels were detected bybiotinylated anti-cystatin C antibody followed by streptavidin-HRP.

FIG. 8A-8C: Effects of NAC therapy in HCCAA patients. Cystatin Cimmunostaining (brown stain) was performed on 3 separate skin biopsiesobtained from the same location of the back, from two members of a HCCAAfamily who are carriers of the hCC L68Q variant, using a rabbit-antihuman cystatin C antibody. The biopsies in each left panel (skin biopsy1 in FIG. 8A and FIG. 8B) were obtained when the family participated inresearch over 2 years prior to the initiation of this work. The biopsiesin the middle figure panels (skin biopsy 2 in FIG. 8A and FIG. 8B) wereobtained approximately 18 months later. The biopsies in the right panels(skin biopsies #3 in FIG. 8A and FIG. 8B) from both subjects showcystatin C protein complex deposition after 6 months of therapy withNAC. A marked reduction was seen in the proband (panel A) and the parent(panel B) after 6 months of NAC therapy. Panel A: Cystatin Cimmunostaining of skin biopsies from the proband. Panel B: Cystatin Cimmunostaining of skin biopsies from the parent. FIG. 8C. Cyst-Cmonomers are detectable at reduced levels in blood from subjectscarrying the L68Q mutation. High molecular weight complexes appear to bepresent in one carrier who is not taking NAC.

DETAILED DESCRIPTION OF THE INVENTION

To create a system in which to test the ability of a compound to impacthCC multimerization while gaining some insight into its toxicity, wecreated cell lines that express high amounts of either wild type ormutant hCC. The cell lines and the monomeric and multimeric hCC thatthey create were characterized and employed in experiments fornon-toxically interfering with aggregation of the mutant protein.Additionally, a biomarker study using NAC to treat human subjects withHCCAA was conducted.

This system facilitates evaluation of the ability of a molecule tointerfere with aggregation of mutant hCC while also providinginformation about toxicity to cells or organisms. Clones of 293T cellsthat overexpress either wild type or mutant hCC were generated. Thesecells produce and secrete detectable levels of hCC. Importantly, we haveestablished conditions that allow detection of high molecular complexesthat form in both lysates and supernatants of cells expressing mutanthCC that are absent in cells expressing comparable amounts of the wildtype protein. We are able to detect the high molecular weight complexesof mutant hCC by Western blotting under non-reducing conditions.Interestingly, a short incubation of either lysate or supernatant withone of two reducing agents, either reduced glutathione (GSH) orN-acetyl-cysteine (NAC), breaks oligomers of the mutant into monomers.Additionally, treatment of L68Q hCC expressing cells with either NAC orGSH reduces oligomerization of secreted hCC L68Q at 24, 48 and 72 h.Patients with HCCAA were subsequently treated with NAC for six months.As a biomarker of response, skin biopsies were obtained to determine ifstaining for amyloid cystatin C complexes were reduced in the skinfollowing treatment. The proband, who was on the highest dose and hadbeen on NAC for 9 months to treat mucous plugs in her lungs and hadpreviously sustained 3 major strokes over a 9 month period prior tostarting NAC, had approximately 75% reduction in the amyloid stain inthe skin and has been event free for the 18 months of NAC therapy.

In summary, this study provides a new cellular model to test newtherapies for the treatment of HCCAA and provides clear evidence thatmutant hCC is a pharmacological target for reducing agents like NAC.Most importantly, the data implicate NAC as a potentially a usefultherapy to treat this devastating disease based on skin biomarkerresults from three patients with HCCAA.

The following definitions are provided to aid in understanding thesubject matter regarded as the invention.

In this invention, “a” or “an” means “at least one” or “one or more,”etc., unless clearly indicated otherwise by context. The term “or” means“and/or” unless stated otherwise. In the case of a multiple-dependentclaim, however, use of the term “or” refers to more than one precedingclaim in the alternative only.

As used herein, “human cystatin C (hCC)” refers to a protein whichfunctions as cysteine protease inhibitor that belongs to the cystatinsuperfamily. hCC is a secretory type 2 cystatin and is expressed in allnucleated human cells. L68Q-hcc refers to a mutated hCC wherein aleucine at position 68 is substituted for a glutamine variant.

The terms “agent” and “test compound” are used interchangeably hereinand denote a chemical compound, a mixture of chemical compounds, abiological macromolecule, or an extract made from biological materialssuch as bacteria, plants, fungi, or animal (particularly mammalian)cells or tissues. Biological macromolecules include siRNA, shRNA,antisense oligonucleotides, peptides, peptide/DNA complexes, and anynucleic acid based molecule which exhibits the capacity to modulate theactivity of the hCC. Example agents include reducing agents such as NACand derivatives thereof used alone and in combination. Other usefulagents include, without limitation, glutathione, monensin, papain,cathepsin B, and falcipain. The biological activity of such agents canbe assessed in the screening assays described herein below.

“Treatment,” as used herein, covers any administration or application ofa therapeutic for disease in a mammal, including a human, and includesinhibiting the disease or progression of the disease, inhibiting orslowing the disease or its progression, arresting its development,partially or fully relieving the disease, preventing the onset of thedisease, or preventing a recurrence of symptoms of the disease. Exampletreatments include administration at least one NAC derivative atefficacious doses.

The terms “inhibition” or “inhibit” refer to a decrease or cessation ofany event (such as fibril formation) or to a decrease or cessation ofany phenotypic characteristic or to the decrease or cessation in theincidence, degree, or likelihood of that characteristic. To “reduce” or“inhibit” is to decrease, reduce or arrest an activity, function, and/oramount as compared to a reference. It is not necessary that theinhibition or reduction be complete. For example, in certainembodiments, “reduce” or “inhibit” refers to the ability to cause anoverall decrease of 20% or greater. In another embodiment, “reduce” or“inhibit” refers to the ability to cause an overall decrease of 50% orgreater. In yet another embodiment, “reduce” or “inhibit” refers to theability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.

The term “inhibitor” refers to an agent that slows down or prevents aparticular chemical reaction, signaling pathway or other process, orthat reduces the activity of a particular reactant, catalyst, or enzyme.

The terms “patient” and “subject” are used interchangeably to mean amammal, including human.

N-acetyl cysteine (NAC)” is a derivative of cysteine that acts to reducedisulphide bonds associated with fibril formation present inneurodegenerative disorders such as HCCAA and Alzheimer's disease. WhileNAC and ester derivatives are exemplified herein, other NAC derivativesare known in the art and described in the following patent documents;U.S. Pat. Nos. 3,242,052, 3,591,686, 3,647,834, 3,749,770, 4,016,287,4,132,803, 4,276,284, 4,331,648, 4,708,965, 4,711,780, 4,721,705,4,724,239, 4,827,016, 4,859,653, 4,868,114, 4,876,283, DE150694C,EP0219455A2, EP0269017A2, EP0280606A1, EP0304017A2, and EP0339508A1which are incorporated herein by reference.

“Nucleic acid” or a “nucleic acid molecule” as used herein refers to anyDNA or RNA molecule, either single or double stranded and, if singlestranded, the molecule of its complementary sequence in either linear orcircular form. In discussing nucleic acid molecules, a sequence orstructure of a particular nucleic acid molecule may be described hereinaccording to the normal convention of providing the sequence in the 5′to 3′ direction.

With reference to nucleic acids of the invention, the term “isolatednucleic acid” is sometimes used. This term, when applied to DNA, refersto a DNA molecule that is separated from sequences with which it isimmediately contiguous in the naturally occurring genome of the organismin which it originated. For example, an “isolated nucleic acid” maycomprise a DNA molecule inserted into a vector, such as a plasmid orvirus vector, or integrated into the genomic DNA of a prokaryotic oreukaryotic cell or host organism.

When applied to RNA, the term “isolated nucleic acid” refers primarilyto an RNA molecule encoded by an isolated DNA molecule as defined above.Alternatively, the term may refer to an RNA molecule that has beensufficiently separated from other nucleic acids with which it would beassociated in its natural state (i.e., in cells or tissues). An isolatednucleic acid (either DNA or RNA) may further represent a moleculeproduced directly by biological or synthetic means and separated fromother components present during its production.

A “replicon” is any genetic element, for example, a plasmid, cosmid,bacmid, phage or virus, that is capable of replication largely under itsown control. A replicon may be either RNA or DNA and may be single ordouble stranded.

A “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage orvirus, to which another genetic sequence or element (either DNA or RNA)may be attached so as to bring about the replication of the attachedsequence or element. Exemplary vectors of the invention include withoutlimitation, adenoviral-based vectors, adeno-associated viral vectors andretroviral vectors.

An “expression operon” refers to a nucleic acid segment that may possesstranscriptional and translational control sequences, such as promoters,enhancers, translational start signals (e.g., ATG or AUG codons),polyadenylation signals, terminators, and the like, and which facilitatethe expression of a polypeptide coding sequence in a host cell ororganism.

The term “isolated protein” or “isolated and purified protein” issometimes used herein. This term refers primarily to a protein producedby expression of an isolated nucleic acid molecule of the invention.Alternatively, this term may refer to a protein that has beensufficiently separated from other proteins with which it would naturallybe associated, so as to exist in “substantially pure” form. “Isolated”is not meant to exclude artificial or synthetic mixtures with othercompounds or materials, or the presence of impurities that do notinterfere with the fundamental activity, and that may be present, forexample, due to incomplete purification, addition of stabilizers, orcompounding into, for example, immunogenic preparations orpharmaceutically acceptable preparations.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight of a given material (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90-95% by weightof the given compound. Purity is measured by methods appropriate for thegiven compound (e.g. chromatographic methods, agarose or polyacrylamidegel electrophoresis, HPLC analysis, and the like).

The term “tag,” “tag sequence” or “protein tag” refers to a chemicalmoiety, either a nucleotide, oligonucleotide, polynucleotide or an aminoacid, peptide or protein or other chemical, that when added to anothersequence, provides additional utility or confers useful properties,particularly in the detection or isolation, to that sequence. Thus, forexample, a homopolymer nucleic acid sequence or a nucleic acid sequencecomplementary to a capture oligonucleotide may be added to a primer orprobe sequence to facilitate the subsequent isolation of an extensionproduct or hybridized product. In the case of protein tags, histidineresidues (e.g., 4 to 8 consecutive histidine residues) may be added toeither the amino- or carboxy-terminus of a protein to facilitate proteinisolation by chelating metal chromatography. Alternatively, amino acidsequences, peptides, proteins or fusion partners representing epitopesor binding determinants reactive with specific antibody molecules orother molecules (e.g., flag epitope, c-myc epitope, transmembraneepitope of the influenza A virus hemaglutinin protein, protein A,cellulose binding domain, calmodulin binding protein, maltose bindingprotein, chitin binding domain, glutathione S-transferase, and the like)may be added to proteins to facilitate protein isolation by proceduressuch as affinity or immunoaffinity chromatography. Chemical tag moietiesinclude such molecules as biotin, which may be added to either nucleicacids or proteins and facilitates isolation or detection by interactionwith avidin reagents, and the like. Numerous other tag moieties areknown to, and can be envisioned by, the trained artisan, and arecontemplated to be within the scope of this definition.

As used herein, the terms “reporter,” “reporter system”, “reportergene,” or “reporter gene product” shall mean an operative genetic systemin which a nucleic acid comprises a gene that encodes a product thatwhen expressed produces a reporter signal that is a readily measurable,e.g., by biological assay, immunoassay, radioimmunoassay, or bycolorimetric, fluorogenic, chemiluminescent or other methods. Thenucleic acid may be either RNA or DNA, linear or circular, single ordouble stranded, antisense or sense polarity, and is operatively linkedto the necessary control elements for the expression of the reportergene product. The required control elements will vary according to thenature of the reporter system and whether the reporter gene is in theform of DNA or RNA, but may include, but not be limited to, suchelements as promoters, enhancers, translational control sequences, polyA addition signals, transcriptional termination signals and the like.

The terms “transform”, “transfect”, “transduce”, shall refer to anymethod or means by which a nucleic acid is introduced into a cell orhost organism and may be used interchangeably to convey the samemeaning. Such methods include, but are not limited to, transfection,electroporation, microinjection, PEG-fusion and the like.

The introduced nucleic acid may or may not be integrated (covalentlylinked) into nucleic acid of the recipient cell or organism. Inbacterial, yeast, plant and mammalian cells, for example, the introducednucleic acid may be maintained as an episomal element or independentreplicon such as a plasmid. Alternatively, the introduced nucleic acidmay become integrated into the nucleic acid of the recipient cell ororganism and be stably maintained in that cell or organism and furtherpassed on or inherited to progeny cells or organisms of the recipientcell or organism. In other manners, the introduced nucleic acid mayexist in the recipient cell or host organism only transiently.

A “clone” or “clonal cell population” is a population of cells derivedfrom a single cell or common ancestor by mitosis.

A “cell line” is a clone of a primary cell or cell population that iscapable of stable growth in vitro for many generations.

Methods and Uses for Treating HCCAA and Other NeurodegenerativeDisorders

Encompassed herein are methods of treating HCCAA and otherneurodegenerative disorders in a subject, comprising administering aneffective amount of NAC or a functional derivative thereof. The term“treatment,” as used herein, includes any administration or applicationof a therapeutic for a disease or disorder in a subject, and includesinhibiting the disease, arresting its development, relieving thesymptoms of the disease, or preventing occurrence or reoccurrence of thedisease or symptoms of the disease.

In some embodiments, the treatment methods comprise identifying ordiagnosing a subject as having a genetic alteration in hCC causative ofHCCAA, and administering a NAC or a functional derivative thereof to theidentified or diagnosed subject. In other embodiments, the subject has adifferent disease associated with pathological fibril formation,including but not limited to Alzheimer's disease.

The total treatment dose or doses (when two or more targets are to bemodulated) can be administered to a subject as a single dose or can beadministered using a fractionated treatment protocol, in whichmultiple/separate doses are administered over a more prolonged period oftime, for example, over the period of a day to allow administration of adaily dosage or over a longer period of time to administer a dose over adesired period of time. One skilled in the art would know that theamount of therapeutic agent required to obtain an effective dose in asubject depends on many factors, including the age, weight and generalhealth of the subject, as well as the route of administration and thenumber of treatments to be administered. In view of these factors, theskilled artisan would adjust the particular dose so as to obtain aneffective dose for treating an individual having HCCAA.

The effective dose of therapeutic agent(s) will depend on the mode ofadministration, and the weight of the individual being treated. Thedosages described herein are generally those for an average adult butcan be adjusted for the treatment of children. The dose will generallyrange from about 0.001 mg to about 1000 mg.

In an individual suffering from a more severe form of the disease,administration of therapeutic agents can be particularly useful whenadministered in combination, for example, with a conventional agent fortreating such a disease. The skilled artisan would administertherapeutic agent(s), alone or in combination and would monitor theeffectiveness of such treatment using routine methods such asneurological or pulmonary function determination, radiologicorimmunologic assays, or, where indicated, histopathologic methods.

Administration of the pharmaceutical preparation is preferably in an“effective amount” this being sufficient to show benefit to theindividual. This amount prevents, alleviates, abates, or otherwisereduces the severity of HCCAA symptoms in a patient. Treatment ofpatients having HCCAA with an efficacious amount of NAC or a functionalderivative thereof may produce improvements in neurological function,respiratory function, tapering of concomitant medication usage, orincreased survival.

The pharmaceutical preparation is formulated in dosage unit form forease of administration and uniformity of dosage. Dosage unit form, asused herein, refers to a physically discrete unit of the pharmaceuticalpreparation appropriate for the patient undergoing treatment. Eachdosage should contain a quantity of active ingredient calculated toproduce the desired effect in association with the selectedpharmaceutical carrier. Procedures for determining the appropriatedosage unit are well known to those skilled in the art.

Dosage units may be proportionately increased or decreased based on theweight of the patient. Appropriate concentrations for alleviation of aparticular pathological condition may be determined by dosageconcentration curve calculations, as known in the art.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in parenteral, oral solid andliquid formulations, subcutaneously, intradermally, intramuscularly,sublingually, topically, intraperitoneal, nasally, percutaneous,respiratory, ophthalmic, suppository, aerosol, topical or other knownroutes of administration. In addition to the agent(s) useful fortreating a HCCAA, the pharmaceutical compositions may containpharmaceutically-acceptable carriers and other ingredients known toenhance and facilitate drug administration. Thus, such compositions mayoptionally contain other components, such as adjuvants, e.g., aqueoussuspensions of aluminum and magnesium hydroxides, and/or otherpharmaceutically acceptable carriers, such as saline. Other possibleformulations, such as nanoparticles, liposomes, resealed erythrocytes,and immunologically based systems may also be used to deliver/administerthe appropriate agent to a patient according to the methods of theinvention. The use of nanoparticles to deliver such agents, as well ascell membrane permeable peptide carriers that can be used are describedin Crombez et al., Biochemical Society Transactions v35:p44 (2007).

The pharmaceutical compositions can also comprise anti-inflammatoryagents for co administration to further alleviate symptoms of amyloiddisease. These include, without limitation, corticosteroids, aspirin,celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin,ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam,salsalate, sulindac, tolmetin, interleukin (IL)-1 receptor antagonist,IL-4, IL-6, IL-10, IL-11, IL-13, cytokine receptors for IL-1, tumornecrosis factor-alpha, IL-18 and derivatives and biosimilars thereof.

The following materials and methods are provided to facilitate thepractice of the present invention.

Cells and hCC WT vs. L68Q Variant Expression Constructs

Human embryonic kidney 293 (HEK-239T) cells were obtained from ATCC(Manassas, Virginia) and grown at 37 ° C. in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% fetal bovine serum. A plasmidcontaining a cDNA of CST3 was obtained from Dharmacon (Lafayette,Colo.). The full length coding sequence was amplified with a c-terminalMyc tag by PCR using the forward primerGATCGAATTCGCCACCATGGCCGGGCCCCTGCGCG (SEQ ID NO: 1) and reverse primerTCGCGGCCGCCTACAGATCCTCTTCTGAGATGAGTTTTTGTTCGGCGTCCTGACAGGTGGATTTCG (SEQID NO: 2) and ligated into the EcoRI and Notl sites of pBABE-CMV-Puro.⁵⁸The L68Q mutation was introduced by QuikChange site-directed mutagenesis(Agilent, Santa Clara, Calif.) using primers:GTGAACTACTTCTTGGACGTCGAGCAGGGCCGAACCACGTGTACC (SEQ ID NO: 3) andGGTACACGTGGTTCGGCCCTGCTCGACGTCCAAGAAGTAGTTCAC (SEQ ID NO: 4). Allsequences were confirmed by Sanger sequencing. Wild type and mutantconstructs were transfected into HEK-293T cells using Fugene HD(Promega, Madison, Wis.), with 3μg DNA and 9 μl of the transfectionreagent, according to the manufacturer's protocol. After transfectioncells were incubated with fresh medium containing puromycin (1 μg/ml)for 3 weeks. After selection, stable clones of each transfectant weregenerated by limiting dilution. Clones were screened by Western blotusing anti-hCystatin C antibody MAB1196 (R&D, Minneapolis, Minn.).

Western Blot

HEK-293T cells expressing hCC WT or the L68Q variant were washed twicewith ice-cold phosphate-buffered saline (PBS) and lysed on ice using afreshly prepared ice-cold cell lysis buffer containing 50 mM Tris-HCl,pH 7.4, 100 mM NaCl, 50 mM β-glycerophosphate, 10% glycerol (w/v), 1%NP-40 (w/v), 1mM EDTA, 2 mM NaVO₄, and a complete, EDTA-free, proteininhibitor cocktail (Roche Applied Science, Mannheim Germany) at 20 μlper mL of lysis buffer. After clearing the cell lysates bycentrifugation (10 minutes, 21,000×g, 4° C.), the supernatants werecollected and used for Western blotting. Sample buffer containing SDS,glycerol, Tris-HCl pH 6.8, and bromophenol blue was added to each sampleto the following final concentrations: 2% SDS, 10% glycerol, 50 mMTris-HCl, 0.02% bromophenol blue. In samples that were reduced, eitherDTT (50 mM final concentration) or β-mercaptoethanol (5% finalconcentration) were added. Equal volumes of lysate or supernatantsamples were loaded on NuPAGE 4-12% Bis-Tris gels (Thermo FisherScientific, Waltham Mass.) without heating/boiling. Proteins weretransferred to PVDF membranes (Millipore, Billerica, Mass.) and blottedwith anti-hCystatin C, and developed by enhanced chemiluminescence (ECL;Thermo Fisher Scientific). ECL films were scanned, and densities ofbands were determined using the gel analysis features of Fiji.⁵⁹

Drug Treatments

HEK-239T cells were plated on 6-well plates and cultured for 2 days, atwhich point reduced glutathione (GSH) (Sigma, St. Louis, Mo.) orN-acetylcystein (NAC) (Sigma) were added to the indicatedconcentrations. Cells were incubated with compounds for 72 hours, and100 μl samples of supernatants were removed at 24, 48, and 72 hours.Supernatants were cleared by centrifugation (10 minutes, 21,000×g, 4°C.). Sample buffer containing SDS, glycerol, Tris-HCl pH 6.8, andbromophenol blue was added to each sample to the following finalconcentrations: 2% SDS, 10% glycerol, 50 mM Tris-HCl, 0.02% bromophenolblue. When indicated, cells were washed with PBS and lysed and cystatinC levels were determined by means of Western blot analysis.

Statistical Analysis

The means and standard deviations of data were calculated. One-sidedT-test tests were used to determine the level of significance withrespect to the untreated samples, with p<0.05 being consideredstatistically significant.

Treatment of HCCAA Patients with NAC

Three 4 mm skin biopsies from the back were taken from each of the threestudied individuals. The skin biopsies were formalin-fixed andparaffin-embedded. They were cut into 3 μm sections forimmunohistochemistry and immunostained with rabbit polyclonal cystatin Cantibody (Sigma, HPA013143) using the EnVision Detection System aspreviously described.²² hCC immunoreactivity in the carriers skinbiopsies was quantified by semi-automated image analysis using theImageJ software as previously described.²² Bright-field images of eachsection from the carriers were captured using a ×20/0.3NA objective. RGBcolor images of the sections were imported to ImageJ. On each image, arectangular 2000×2000 pixels region of interest (ROI) was defined.Subsequent processing yielding the % area coverage of hCCimmunoreactivity within each ROI was performed as previouslydescribed²². The first biopsy was a historical biopsy takenapproximately 2 years before the beginning of the study, during whichthe proband had experienced over 9 months of NAC therapy (400 mg 4× perday) to treat mucus plugging in the lungs following her third stroke.The second biopsy was taken immediately prior to the family-wideinitiation of NAC treatment (600 mg of NAC 3× per day for 6 months). Thethird biopsy was taken following 6 months of 600 mg 3× per day of NACtherapy.

The proband received 400 mg NAC 4× per day for 9 months followed by 600mg 3× per day for 6 months. The parent received only the 600 mg 3× perday for 6 months course. The proband never missed a dose; the parent didmiss the middle dose 2-3 times per week.

Study Approval

All necessary permits for the use of skin biopsies from L68Q-CST3carriers, and records associated with samples as well as medicalinformation, were obtained from the National Bioethics Committee ofIceland, reference numbers 04-046-S2 and 15-060-S1. Both family memberssigned the informed consent. The NAC therapy was clinically andserendipitously prescribed as a mucolytic therapy to treat lungatelectasis in the proband. The other family member took NAC as adietary supplement (i.e., purchasing NAC on-line through Amazon).

The following Examples are provided to illustrate certain embodiments ofthe invention. It is not intended to limit the invention in any way.

EXAMPLE I

As discussed above, HCCAA is a dominantly inherited disease caused by aleucine 68 to glutamine variant of Human Cystatin C (hCC; L68Q-hCC)(reference). Most carriers of the mutation suffer micro-infarcts andbrain hemorrhages in their twenties leading to paralysis, dementia anddeath in young adults, with an average live expectancy of 30 years(1-5). Post-mortem studies in humans show that hCC was deposited in allbrain areas, grey and white matter alike, most prominently in arteriesand arterioles. These deposits are composed of amyloid fibers,consisting of hCC; this can be demonstrated through staining of postmortem tissue with Congo red staining which causes amyloid structures toshow birefringence under polarized light (6).

To create a system in which to test the ability of a compound to impacthCC multimerization while gaining some insight into its toxicity, wecreated cell lines that express high amounts of either wild type ormutant hCC. This example describes our characterization of the celllines and the monomeric and multimeric hCC that they create, attempts tonon-toxically interfere with aggregation of the mutant protein as wellas a pilot biomarker study using NAC to treat human subjects with HCCAA.

Genetically Engineered HEK-293T Cells Produce and Secrete hCC (Wt orL680) Capable of Oligomerizing Under Non-Reducing Conditions

In order to identify therapeutics agents capable of stopping theproduction of oligomers and fibrils of L68Q hCC, we generatedgenetically engineered HEK-293T cells with the expression of either wildtype (WT) or L68Q mutant hCC. The protein was tagged at the c-terminuswith a myc-tag; c-terminal tagging was chosen to avoid any interferencewith the cleavage of the signal peptide or secretion of the producedprotein that may result from n-terminal tagging. After stableintegration of these constructs into HEK-293T cells, we monitored boththe secreted and the intracellular steady state levels of hCC WT and theL68Q variant. Analysis of hCC was developed by an SDS-PAGE gelelectrophoresis system that allows the formation and detection of low-and high-molecular weight oligomer (LMW and HMW). As shown in FIG. 1A,cells produce and secrete detectable levels of both, hCC WT or thevariant L68Q, capable of oligomerizing under non-reducing conditions(lanes #1 and 3). WT and L68Q expressing cells contain similar levels ofhCC protein in lysates, indicating similar expression levels. However,conditioned supernatants from the L68Q expressing cells contain far lesshCC protein than supernatants from the WT expressing cells. Thisindicates that the L68Q variant protein is not secreted from cells aseffectively as the WT, consistent with previous reports (17, 18). Whileintracellular hCC WT exists predominantly as a monomer, with a smallpercentage of dimer (99 and 1%, respectively), intracellular hCC L68Qvariant was found forming monomer, dimer but also LMW and HMW species asexpected due to its increased propensity to form oligomers (19).Interestingly, the secreted form of hCC WT behaves similarly to theintracellular fraction, being found mainly as a monomer. In comparison,secreted hCC L68Q is found only as HMW. Remarkably, the oligomerizationof both proteins, WT and L68Q variant, are completely abolished inpresence of reducing agents DTT or b-mercaptoethanol; both are strongreducing agents that cause reduction of a typical disulfide bond.

Immunofluorescence assay was performed to detect the level of hCCprotein in untransfected or WT and L68Q expressing 293T cells. hCCprotein was mainly expressed in the cytoplasma. It shows a subcellulardistribution consistent with the previously reported localization inlate-endosomes/prelysosomes, as well as in the Golgi/ER/early-endosomalcompartment, the latter in large agreement with typical characteristicsof a secretory protein (20).

Incubation with Glutathione Impairs hCC Di/Oligomerization in CellularExtracts and Supernatants

Depletion of LMW and HMW in presence of DTT or β-mercaptoetanolhighlights the importance of disulfide bonds for thedimerization/oligomerization process; therefore we hypothesize thattreatment with other reducing agents will impair the dimerization. Weextensively characterized the effect of reducing agents on thedimerization/oligomerization levels of both the secreted and theintracellular levels of hCC WT and L68Q variant. Supernatants andcellular extracts were treated with different concentrations of GSH at37° C. for 15 min. Notably, as FIG. 2A shows, treatments with 3 or 10 mMof GSH severely reduced the amount of dimer and/or HMW oligomer observedin both the secreted and the intracellular fraction of hCC WT or theL68Q variant. Quantitation of these results by densitometry shows that 3mM of GSH displayed a ≈90% inhibition of the HMW in the secretedfraction and ≈50% inhibition on the intracellular fraction of the L68QhCC variant (FIG. 2A and FIG. 1B).

Incubation with NAC or Gluthathione Impairs Dimerization of Secreted hCCL680

Oxidized/reduced glutathione pair is critical to fight against oxidativestress, and, as shown in FIG. 2, it can effectively disrupt the dimersand HMW oligomers of hCC. Accordingly, we analyzed whether anotherreducing agent, the commonly used dietary supplement NAC (with similarantioxidant effects as GSH) would affect theoligomerization/dimerization of secreted hCC. Supernatants were treatedwith different concentrations of GSH and NAC at 37° C. for 60 min. AsFIG. 3A shows, treatments with 3 or 10 mM of glutathione or NAC severelyreduces the oligomerization/dimerization levels of secreted hCC L68Qvariant in vitro. Quantitation showed almost complete ablation of HMWwith 3 mM concentration of either GSH or NAC (FIG. 3A and FIG. 2B). Thisresult clearly demonstrates that GSH or NAC are able to decrease theoligomerization levels of the pathogenic version of hCC L68Q and can bepotentially used as for treatment of patients HCCAA.

Presence of GSH or NAC Reduces Oligomerization of secreted Cystatin CL680 at 24, 48 and 72 h

To investigate whether the effect of NAC or GSH reduces theolygomerization of secreted hCC L68Q in a cellular system morereflective of in vivo biology, we treated cells expressing hCC WT orL68Q with both agents. Cells were seeded in plates and allowed tosecrete hCC for 48 hours, at which point increasing concentrations ofGSH or NAC were added to the cultures. Cells were cultured for 72 hoursin the presence of reducing agents, with samples of the supernatantsbeing removed after 24, 48, and 72 hours. Oligomerization status of hCCwas determined by western blot at each time point. Cells were viable forthe duration of the experiment in the presence of all concentrations (upto 10 mM) of both reducing agents. Proliferation of the cells wasslightly impacted at the highest 10 mM concentration (data not shown).As shown in FIG. 4 (and FIG. 3B), treatment of cells with 10 mM ofeither GSH or NAC completely abolished the presence of HMW and LMW at 24h and 48 h time points, and appreciable but incomplete reduction of HMWand LMW persisted at 72 h. Treatments with lower doses of NAC or GSHwere only incompletely effective at 24 h and 48 h and no significanteffect was detected after 72 h.

It is clear that treatment with reducing agents such as NAC or reducedglutathione of either supernatants or cellular extracts from cell linesengineered to overexpress the mutant version of human cystatin C(Cyst-C) reduces the formation of high molecular complexes of L68Qmutant Cyst-C. To determine if the effects of NAC and GSH are due totheir capacity as reducing agents or some other properties of thecompounds, supernatants and cell extracts were treated with compoundsstructurally similar to NAC and GSH that lack reducing activity. Asshown in FIG. 5, treatment with either n-acetyl-serine (NAS), where thereducing sulfhydryl group in NAC is replaced with a hydroxyl group, orthe oxidized form of glutathione (GSSH), does not affect high molecularweight complexes of L68Q Cyst-C, while significant reduction wasobserved with both reducing agents. The reducing activity of either NACor GSH is required for the effects on Cyst-C oligomerization.

Multiple derivatives of NAC exhibiting improved reducing activity andbioavailability, as well as the ability to cross the blood/brain barrierhave been generated. Two NAC derivatives have been tested with our cellculture system. As shown in FIG. 6, both an amide- and a methyl-esterderivative of NAC retain the ability to disrupt high molecular weightcomplexes of L68Q Cyst-C, when either supernatants or cell extracts aretreated in vitro. Our data also indicate that both derivatives may beslightly more potent in their ability to disrupt oligomerization, asloss of high molecular signal was observed at 1 mM of the derivativesequivalent to that seen with 10 mM of NAC.

Additional results have been generated from transgenic mice that wereobtained from our collaborator Eufrat Levy at New York University. Thesemice have been transformed with a human genomic DNA which contains thecoding sequence of Cyst-C, while lacking non-coding portions of the genewhich may impact expression levels. The mice do not display a phenotypecomparable to that of HCCAA. However, we as shown in FIG. 7, we havebeen able to show the presence of high molecular weight Cyst-C complexesin both brain and blood of transgenic animals. Western blots from themouse tissue extracts are not as clean as blots from the cell system, asthe antibody we use to detect was raised in a mouse. Despite thiscomplication, comparison of the transgenic mice (numbers 6028 and 6019)to the non-transgenic C57B16 animal shows considerable signal caused bythe transgenic human Cyst-C. Although there are several non-specifichigh molecular weight bands observed in the non-transgenic samples, aclear high molecular weight “smear” is seen in the transgenic animals,consistent with what we observe in supernatant from the cell culturesystem. Treatment with NAC reduces this smear, and causes the appearanceof monomer, showing that NAC is capable of reducing oligomerization inbiological samples.

Effects of NAC Therapy in HCCAA Patients

There are several hundred patients in Iceland who suffer from HCCAA(i.e., suffering major strokes in their early 20's) and they all resultfrom a founder mutation from the early 1500s. We have performed RNAseqon 30 subjects from 3 multiplex families and shown that genes involvedin coronary disease, stroke and atherosclerosis are upregulated inmutation carriers of Cystatin C. As there is no therapy available forthese patients, intervention that has a potential to delay or reversethe disease process would be readily approved by the Icelandic MedicinalAgency. Amyloid fiber dimerization is a critical step in the amyloiddeposition process into small-medium sized brain arteries. In cell-basedassays, we have shown that both the wild type and mutated proteins areexpressed and that expression of the mutated protein dimerizes, aprocess that can be inhibited. Thus, drugs that block dimerization ofthe amyloid fibers would be anticipated to be effective in preventingamyloid deposition and halt progression of the disease process, therebypresenting an effective therapy.

FIG. 8A demonstrates the changes in staining from biopsy 1 obtained inall 3 individuals, 2 years ago, biopsy 2 obtained 6 months ago andbiopsy 3 obtained 2 weeks ago post 6 months of NAC therapy. So overallthe drug is reducing the intensity of the biomarker (amyloid-cystatinprotein aggregate) in the skin, suggesting that amyloid precipitation inother organs is also reduced as previously demonstrated (21).

Based on staining results measuring the amyloid-cystatin protein complexaggregates in the skin, it became evident that the proband who had veryhigh level of amyloid-cystatin stain on the first skin biopsy had notprogressed in any significant way (measured by the intensity of thestain) between skin biopsy #1 and #2, whereas her father and her oldersibling (both of whom are also carriers of the L68Q mutant) showedsignificant progression in the intensity of the stain, reflective ofincreased amyloid complex precipitation in the skin over time in theabsence of NAC therapy. It is noteworthy that the proband was on the NACdrug for about 9 months to treat her lungs. She had stopped the therapyonly for a few months prior to the second biopsy. Second biopsy wasperformed first as a baseline to serve as biomarker response tosubsequent NAC therapy. The three biopsies for each individual werestained all together at the same time, for legitimate comparison. Thelead proband (stroke×3 in 9 months) has been 100% compliant with NACtherapy of 600 mg 3× per day and she demonstrated highly visiblereduction in the amyloid stain in comparison with her original skinbiopsy, which amounted to 75% reduction at the end of the 6 months ofprospective therapy (FIG. 8A). Her father's reduction in stainingamounted to 50% and reduction in staining of her sister's biopsy wasless obvious as she had taken lower doses of NAC as indicated inmaterial and methods sections.

Finally, blood samples were acquired from 7 members of an Icelandicfamily known to be carriers of the L68Q mutation. Five of the familymembers were of known mutation status (3 L68Q carriers, 2 wild type),and DNA from all individuals was Sanger sequenced to confirm knownstatuses and to determine the status of the previously unexaminedindividuals, one of whom was found to carry the mutation. Mutationstatus and relationship to the proband in this family are shown in FIG.8B. Western blotting of plasma run under reducing conditions showed areduction of the amount of total Cyst-C in adult carriers of the L68Qmutation (proband, sibling, father). The child carrier did not show areduction in the amount of protein, indicating potential age-relatedeffects (also not on any therapy). Blotting of non-reduced samplesshowed detection of high molecular weight complexes in the childcarrier. In other subjects, interpretation of these results iscomplicated by the fact that the adult carriers of the mutation are alltaking NAC regularly. It is possible that oligomers would be detectablein adult L68Q carriers not taking NAC.

Both NAC derivatives have been proposed to have increased membranepermeability, due to the replacement of a hydroxyl group with less polarsubstituents. Increased membrane permeability often correlates withbetter crossing of the blood brain barrier. To access the membranepermeability of the derivative compounds, live cells were treated withNAC or the derivatives. If the compounds cross the cell membrane, weexpect to see impacts of the derivatives on accumulation ofintracellular oligomers of L68Q Cyst-C. As shown in FIG. 6, thecompounds reduced the amount of high molecular weight Cyst-C in thesupernatants. The lesser effects on the intracellular material is likelydue to a timing issue. The cells continuously produce L68Q Cyst-C atoverexpressed levels, and any compound that enters the cells may beconsumed quickly, having an earlier effect that is lost with continuedculture.

DISCUSSION

Identification of agents with the ability to reduce hCC dimerization andamyloid fibril formation is the key for the development of drugs for thetreatment and/or prevention of the amyloid formation and lethal brainhemorrhage associated with HCCAA. The hCC variant is responsible forHCCAA and there is no treatment available to avoid early death by brainhemorrhage. Here, we first created cells that produce and secretedetectable levels of hCC (wt or L68Q) capable of oligomerizing undernon-reducing conditions, and we then show that short incubation witheither GSH or NAC breaks oligomers into monomers of both intracellularand secreted hCC L68Q. We show that treatment with either NAC or GSHreduces oligomerization of the secreted hCC L68Q at 24, 48 and 72 h andthat treatment with NAC in human patients not only prevents ongoingprecipitation of amyloid in the skin, but also reduces previouslyprecipitated amyloid in a significant way, with over 75% reductionobserved following 6 months of oral therapy with doses that are welltolerated and without adverse events.

The cellular system developed was constructed to identify agents thatcould reduce the hCC dimerization and amyloid fibril formation “in vivo”of both wt cystatin C and L68Q variant. Previous systems for the studyof the dimerization of hCC had been developed, however, most of themwere performed mainly with wildtype cystatin C as it is extremelydifficult to produce sufficient amounts of monomeric L68Q-cystatin C(14). The genetically engineered HEK-293T cells with the expression ofboth c-terminal tagged wt and L68Q hCC provide a superior model to studyand characterize the impact of the small molecules on both the secretedand the intracellular levels of wt and L68Q hCC. It is important tohighlight that the study and characterization of agents that reduceoligomerization should be made on both fractions because the behavior isdifferent; in particular, on the L68Q-hCC variant. This variant wasfound mainly as LMW oligomers in the intracellular fraction but mainlyforms HMW in the extracellular compartment. These can be due to eitherthe secretion process induces oligomerization of the L68Q variant orbecause the environmental conditions of the extracellular compartmentpromote the oligomerization of this variant or because theoligomerization prolonges the half-life of the protein.

L68Q cystatin C, is highly amyloidogenic, and subjects carrying thecorresponding mutation suffer from massive cerebral amyloidosis leadingto brain hemorrhage and death in early adult life (16). Other amyloiddiseases such as Alzheimer, Parkinson, and HD have similar amyloidorigins and they are also caused by accumulation of misfolded proteins.This broad-spectrum effect of proteotoxic stress has led to the term“proteinopathies” for neurodegenerative diseases. Interestingly, therisk of getting any of these neurodegenerative diseases increasesdramatically with age (22), probably, as a consequence of an increase inprotein-misfolding stress, a reduced proteasome activity and a decreasein antioxidant defenses that drive to an extracellular accumulation ofmisfolded proteins (22). The proteasome and autophagy-lysosomal pathwaysare the major routes for intracellular aggregation clearance. However,little is known about any corresponding mechanisms that operateextracellularly and about effective strategies to slow or prevent theneurodegeneration resulting from these diseases in humans (23).

Glutathione (GSH) is synthesized in the cytosol from the precursor aminoacids glutamate, cysteine and glycine and it is considered the primaryendogenous antioxidant in the cell. It is present in the cell atdifferent concentrations, which can go up to 10 mM, depending on thesubcellular compartment being present at high concentrations on thecytosol and very low concentration inside the ER (24). Protein disulfidebonds rarely form in the cytosol because of the high concentrations ofGSH, by contrast, the lumen of the endoplasmic reticulum (ER) and theextracellular compartment contains a relatively higher concentration ofoxidized glutathione (GSSG) (25). This differential distribution of GSHallows the formation of native disulfide bonds in the ER through acomplex process involving not only disulfide-bond formation, but alsothe isomerization of non-native disulfide bonds. Our immunofluorescencestudies and previous reports indicate that hCC localizes inlate-endosomes/prelysosomes, as well as in the Golgi/ER/early-endosomalcompartment (20). This localization is in agreement with typicalcharacteristics of a secretory protein and it is consistent with thehypothesis that L68Q hCC polymerizes in these compartments whereexposure to GSH is reduced thereby increasing aggregation, thus,explaining the released aggregates in the extracellular compartment.

Under normal conditions, GSH levels are regulated by two majormechanisms: by controlling the rates of its synthesis and of its exportfrom cells; however, GSH levels are also influenced by agents orconditions that alter the thiol redox state that lead to the formationof glutathione S-conjugates or complexes, and/or that disrupt thedistribution of GSH among various intracellular organelles. In addition,GSH levels are affected by the nutritional status and hormonal/stresslevels, they exhibit developmental and diurnal variations, and areaffected by certain physiological states, including pregnancy andexercise (26-33). Physiological levels of GSH in blood should provide anappropriate antioxidant environment that avoids extracellularaccumulation of proteins, however, presence of mutations like hCC L68Qor deficiencies in the levels of GSH, as a consequence of nutritionalstatus or age, could drive to undesired accumulation of misfoldedproteins (3). In addition, it is known that GSH deficiency, or adecrease in the GSH/glutathione disulfide (GSSG) ratio, manifests itselflargely through an increased susceptibility to oxidative stress, and theresulting damage is thought to be involved in diseases such asParkinson's disease, and Alzheimer's disease and it is stronglyassociated with other age-related pathologies (34, 35). Results shown inthis work indicates that NAC can represent an interesting therapeuticapproach to amyloid diseases such as HCCAA, by the reduction ofaccumulation of amyloid proteins.

Acetylcysteine is a synthetic N-acetyl derivative of the endogenousamino acid L-cysteine, a precursor of the antioxidant enzymeglutathione. Both GSH and NAC already have been approved for use inhumans and have been administered at high doses for long periods withoutadverse side effects. They work as a direct reactive oxygen species(ROS) scavenger and as a source of SH groups, stimulating the GSHsynthesis and increasing the presence of 1) non-protein and 2) proteinSH groups. Acetylcysteine, in addition, also regenerates liver stores ofGSH. These effects confer NAC the ability to reduce disulfide bounds andare the reason why NAC is widely used to reduce viscosity and elasticityof the mucus among other uses. Our data show that treatment withantioxidants such as GSH and NAC (and also DTT or beta-MetOH) abolisheshCC oligomerization. This effect indicates that disulfide bond formationis essential for the oligomerization process. Disulfide bonds do notappear to be directly involved in the dimerization process (16), howeverthe presence of two disulfide bonds in human cystatin C (as in all type2 cystatins), and the preservation of them in the dimeric structureindicates its key role in the dimerization process (16). We postulatethat the intramolecular disulfide bonds are essential for the correctfolding of the hCC monomer and for the exchange of three-dimensional‘subdomains’ between the two subunits of the dimer and its impairmentabolish the oligomerization.

Our data show that treatment with NAC will increase GSH production andboth antioxidants will reduce oligomerization of the secreted hCCreducing the amyloid formation on the brain of persons with HCCAA.Treatment with GSH may be effective, however, its low bioavailabilitylimits it's potential as a therapeutic for the treatment of patientswith HCCAA. NAC appears as the perfect candidate because of its role inrestoring GSH levels, antioxidant properties, and its ability to breakdisulfide bonds reviewed in (36). In addition, NAC supplementationsignificantly improved coronary and peripheral vasodilatation (37).Specific to brain disorders, NAC has been administered with someefficacy in patients with Alzheimer disease (38), and our data show thatit can be a good alternative for the HCCAA.

Cellular membranes, along with the blood-brain barrier, exhibit reducedpermeability to NAC, thus extracellular NAC treatment does not appear toimpact the dimerization status of the intracellular levels of L68Q hCC(data not shown). Accordingly, the effects of NAC derivatives includingwithout limitation, N-acetylcysteine ethyl ester (NACET) orN-acetylcysteine methyl ester are preferably administered. These novellipophilic cell permeable membrane cysteine derivatives should providegood candidates for the oral use as an H2S producer in the treatment ofamyloid disease like HCCAA (39).

The reduction observed in the amyloid stain in the skin biopsies withNAC treatment is highly encouraging and indicates that this therapy willhave efficacy in treating patients with HCCAA. As amyloid precipitatesin all organs, there is no reason to believe that there is ongoingprecipitation and accumulation of amyloid in the brain, when reductionis observed in the skin. More likely, there is similar reduction inother body organs, including brain vessels and the brain. No new eventshave occurred in any of the 3 individuals, all of whom have continuedtherapy and the proband is now approximately two years post her thirdand last stroke.

It is noteworthy that a significant number of the HCCAA patients inIceland never get a clinical stroke, and only present with dementia atan early age. As the disease process of amyloid precipitation iscomparable in HCCAA patients to that in Alzheimer disease, blocking theability of amyloid fibers to dimerize and polymerize (which is enhancedby the L68Q-cystatin C founder mutation cases), could help Alzheimerpatients with amyloid associated dementia. Thus, NAC therapy or NAC-likecompounds could be beneficial for Alzheimer disease.

The analogy here is familial combined hypercholesterolemia (FCH), asstatin drugs were developed to treat this familial condition (patientswith FCH develop stroke and myocardial infarction in their 20s); itsubsequently became evident that elevated cholesterol was harmful and amajor risk factor for MI and stroke, and that patients with CV riskfactors benefitted from statin treatment. HCCAA is an enhanced amyloidprecipitation that occurs in early life and leads to catastrophic eventsin the 20's and early dementia. This process is somewhat comparable butoccurs slower in Alzheimer disease so the dementia typically presentsnot until mid to late 60's or 70's—whereas the treatment would be thesame.

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While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. It will be apparentto one skilled in the art that various changes and modifications can bemade therein without departing from the scope of the present invention,as set forth in the following claims.

1. A method for treating amyloid deposit disease comprising deliveringan effective amount of at least one antioxidant to a patient at risk foramyloid disease. 2-22. (canceled)